Immunoglobulin (Ig) class switch recombination (CSR) is a process by which B cells exchange the constant domain of the Ig heavy chain for the optimal clearance of pathogens. This unique DNA recombination is directed by kilobase- long switch regions and requires B cell-specific factor activation-induced cytidine deaminase (AID) as well as other ubiquitously expressed DNA repair factors. It is known that CSR is initiated by AID-catalyzed cytidine deamination resulting in uracils in the switch regions. However, the mechanism by which switch region directs AID actions in-cis and the interplays of uracil repair pathways that ultimately lead to DNA double strand breaks remain poorly defined. The objectives of this application are to identify cis-acting DNA sequences in the switch region and trans-acting protein factors that are responsible for targeted DNA cleavage at defined genomic loci during class switch recombination. We have developed a cell-based class switch assay for studying the function of switch region sequences at the endogenous chromosomal locus. This assay was based on our recent success in highly efficient gene targeting in CH12F3 cells, a mouse B cell line capable of robust cytokine-induced CSR in vitro. We have designed an efficient knock-in strategy to allow assessment of a large number of switch region mutations. Highly efficient gene targeting in CH12F3 cells also allows study of gene function by the reverse genetic approaches in a cellular model for CSR. We are now in position to address several important yet unanswered questions previously difficult to address in animal models. We propose three specific aims: (1) Identify short sequence motifs required for class switch recombination;(2) Identify long sequence organizations required for class switch recombination;(3) Identify the DNA cleavage activity at switch regions. The completion of this project will lead to a more complete understanding of the function of switch region sequences and the identification of the nucleases involved in DNA cleavage in CSR. These findings will provide mechanistic insight to a variety of human diseases involving class switch recombination. Public Health Relevance: This proposed project focuses on elucidating the molecular mechanism of class switch recombination, a process by which the B cell changes the isotype of the antibody for optimal elimination of pathogens. Understanding the mechanism of class switch recombination is important for biological reasons (a unique regional- specific DNA recombination), immunological reasons (infection, allergy and autoimmunity) and its cancer-relevance (oncogenic chromosomal translocations in a variety of B cell lymphomas).